1. Technical Field
The disclosure relates to an inverted microscope system that irradiates a specimen with illumination light to observe the specimen by receiving the light reflected from the specimen.
2. Related Art
A microscope that illuminates a specimen to observe the specimen has conventionally been used for observing, for example, a cell in the field of medicine, biology, or the like. Furthermore, in the industrial field, a microscope system is used for various purposes such as the quality management of a metal structure, the research development of new materials, or the inspection of electronic devices or magnetic heads.
Examples of an optical system used for observing fluorescence from a specimen include a total internal reflection fluorescence microscopy optical system. In the total internal reflection fluorescence microscopy optical system, illumination light is totally reflected from a glass interface on which a specimen is placed to illuminate only the specimen just near the glass interface using an evanescent wave generated by the total reflection illumination, and thus the generated fluorescence can be observed.
As an exemplary application of the total internal reflection fluorescence microscopy optical system, ion transfer from/into a cell membrane by light stimulation, for example, using channelrhodopsin attracts attention. A local light stimulation using channelrhodpsin can be performed in addition to the illumination observation of only the periphery of the cell membrane of the cell that sticks on the glass.
Here, there is a phenomenon in which channelrhodopsin changes the ionic concentration of the cell sticking on the glass and this puts the cell into an excitatory state, and then the excitation is propagated to cells around the cell. In that case, the cells around the cell exist on upper layers than the cell sticking on the glass from the glass surface. However, the observation range in the total internal reflection fluorescence microscopy optical system is up to several hundred nm from the glass interface within the reach of the evanescent wave. Thus, the phenomenon of the propagation cannot be observed in the total internal reflection fluorescence microscopy optical system.
On the other hand, there is a disk scanning confocal optical system as another optical system used for observing the fluorescence from a specimen. The disk scanning confocal optical system is capable of observing only the fluorescence from the specimen surface in real time by spinning, at a high speed, a disk on which a confocal opening such as a slit or a pinhole is placed at a position conjugate to the specimen surface (objective lens focal position).
In the disk scanning confocal optical system, the specimen is irradiated through the slit or pinhole on the disk and thus only a fluorescent image from a desirable cross-section can be observed in real time. In an observation, for example, of fluorescence resonance energy transfer (FRET), the excitation of the cell can be determined from the color of the fluorescence. Thus, even in an experimental system in which an excitatory state is propagated in the optical axis direction, the effect of sectioning that is a feature of the disk scanning confocal optical system makes it possible to detect the intensity of variations in the excitation state as color variations for each cross-section.
As a technique for performing a local light stimulation on a starting point of a propagation to observe the propagation of the excitatory state of a cell, for example, Japanese Laid-open Patent Publication No. 2005-24647 discloses a microscope that is a combination of a total internal reflection fluorescence microscopy optical system and a disk scanning confocal optical system. In the technique, the total internal reflection fluorescence microscopy optical system is placed below the specimen and the disk scanning confocal optical system is placed above the specimen.